Summary of Work: Exogenous DNA damaging agents such as cisplatinin, nitrogen mustard, and psoralen create interstrand DNA crosslinks. Such non-coding lesions must be repaired to ensure accurate replication of the genome and viability of the organism. The Drosophila mus308 mutation, which confers hypersensitivity to nitrogen mustard but not the monofunctional agent methyl-methane sulfonate, identified a DNA polymerase likely involved in processing DNA crosslinks. Based on homology to the Drosophila mus308 gene and another Family A DNA polymerase, pol g, we previously cloned and expressed the cDNA for human DNA polymerase q. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular weight of ~200 kDa. The C-terminal region contains the canonical DNA polymerase motifs A, B and C found in the family A type of DNA polymerases which include E. coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31. We have overproduced a 100 kDa and 200 kDa protein in E. coli and baculoviral infected cells and purified the recombinant protein. Both polypeptides have the expected DNA polymerase activity. Site-directed alterations of active site residues in the DNA polymerase have been produced to confirm the DNA polymerase function. Recent inspection of the gene indicates a much larger coding region for pol theta, encoding a protein with a molecular weight of 300 kDa, containing motifs for DNA polymerase, exonuclease, and a helicase. Current effort are focused to isolate, clone and overexpress the full length cDNA coding for this 300 kDa protein.